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人柯薩奇病毒 A16（Cox-A16）酶聯(lián)免疫分析（ELISA）
試劑盒使用說明書
本試劑僅供研究使用 目的：本試劑盒用于測定人血清，血漿及相關
液體樣本中柯薩奇病毒 A16（Cox-A16）水平。
實驗原理：
本試劑盒采用雙抗體夾心酶聯(lián)免疫法 （ELISA） 測定標本中人柯薩奇病毒 A16 （Cox-A16） 。
用純化的人柯薩奇病毒 A16（Cox-A16）抗體包被微孔板，制成固相抗體，可與樣品中柯薩
奇病毒 A16（Cox-A16）相結合，經(jīng)洗滌除去未結合的抗原和其他成分后再與 HRP 標記的
柯薩奇病毒 A16（Cox-A16）抗體結合，形成抗體-抗原-酶標抗體復合物，經(jīng)過*洗滌后
加底物 TMB 顯色。TMB 在 HRP 酶的催化下轉化成藍色，并在酸的作用下轉化成zui終的黃
色。用酶標儀在 450nm 波長下測定吸光度（OD 值） ，與 CUTOFF 值相比較，從而判定標本
中人柯薩奇病毒 A16（Cox-A16）的存在與否。
試劑盒組成：
試劑盒組成 48孔配置 96孔配置 保存
說明書 1 份 1份
封板膜 2片（48） 2片（96）
密封袋 1 個 1個
酶標包被板 1×48 1×96 2-8℃保存
陰性對照 0.5ml×1 瓶 0.5ml×1瓶 2-8℃保存
陽性對照 0.5ml×1 瓶 0.5ml×1瓶 2-8℃保存
酶標試劑 3 ml×1 瓶 6 ml×1瓶 2-8℃保存
樣品稀釋液 3 ml×1 瓶 6 ml×1瓶 2-8℃保存
顯色劑 A 液 3 ml×1 瓶 6 ml×1瓶 2-8℃保存
顯色劑 B 液 3 ml×1 瓶 6 ml×1瓶 2-8℃保存
終止液 3ml×1 瓶 6ml×1 瓶 2-8℃保存
濃縮洗滌液 （20ml×20倍）×1瓶 （20ml×30 倍）×1瓶 2-8℃保存
樣本處理及要求：
1. 血清：室溫血液自然凝固 10-20 分鐘，離心 20分鐘左右（2000-3000 轉/分） 。仔細收集上
清，保存過程中如出現(xiàn)沉淀，應再次離心。
2. 血漿：應根據(jù)標本的要求選擇 EDTA或檸檬酸鈉作為抗凝劑，混合 10-20 分鐘后，離心
20 分鐘左右（2000-3000 轉/分） 。仔細收集上清，保存過程中如有沉淀形成，應該再次
離心。
3. 尿液：用無菌管收集，離心 20 分鐘左右（2000-3000 轉/分） 。仔細收集上清，保存過程
中如有沉淀形成，應再次離心。胸腹水、腦脊液參照實行。
4. 細胞培養(yǎng)上清：檢測分泌性的成份時，用無菌管收集。離心 20分鐘左右（2000-3000 轉/
分） 。仔細收集上清。檢測細胞內的成份時，用 PBS（PH7.2-7.4）稀釋細胞懸液，細胞2
濃度達到 100萬/ml 左右。通過反復凍融，以使細胞破壞并放出細胞內成份。離心 20 分
鐘左右（2000-3000轉/分） 。仔細收集上清。保存過程中如有沉淀形成，應再次離心。
5. 組織標本：切割標本后，稱取重量。加入一定量的 PBS，PH7.4。用液氮迅速冷凍保存?zhèn)?br />用。標本融化后仍然保持 2-8℃的溫度。加入一定量的 PBS（PH7.4） ，用手工或勻漿器
將標本勻漿充分。離心 20 分鐘左右（2000-3000 轉/分） 。仔細收集上清。分裝后一份待
檢測，其余冷凍備用。
6. 標本采集后盡早進行提取，提取按相關文獻進行，提取后應盡快進行實驗。若不能馬上
進行試驗，可將標本放于-20℃保存，但應避免反復凍融.
7. 不能檢測含 NaN3的樣品，因 NaN3 抑制辣根過氧化物酶的（HRP）活性。
操作步驟：
1. 編號：將樣品對應微孔按序編號，每板應設陰性對照 2孔、陽性對照 2 孔、空白對照 1
孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）
2. 加樣：分別在陰、陽性對照孔中加入陰性對照、陽性對照 50μl。然后在待測樣品孔先
加樣品稀釋液 40μl，然后再加待測樣品 10μl。加樣將樣品加于酶標板孔底部，盡量不
觸及孔壁，輕輕晃動混勻，
3. 溫育：用封板膜封板后置 37℃溫育 30分鐘。
4. 配液：將 30（48T 的 20倍）倍濃縮洗滌液加蒸餾水至 600ml 后備用
5. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置 30 秒后棄去，如此
重復 5次，拍干。
6. 加酶：每孔加入酶標試劑 50μl，空白孔除外。
7. 溫育：操作同 3。
8. 洗滌：操作同 5。
9. 顯色：每孔先加入顯色劑 A 50μl，再加入顯色劑 B 50μl，輕輕震蕩混勻，37℃避光顯色
15分鐘
10. 終止：每孔加終止液 50μl，終止反應（此時藍色立轉黃色） 。
11. 測定：以空白空調零，450nm波長依序測量各孔的吸光度（OD 值） 。 測定應在加終止
液后 15分鐘以內進行。
結果判定：
試驗有效性：陽性對照孔平均值≥1.00; 陰性對照平均值≤0.10
臨界值（CUT OFF）計算：臨界值=陰性對照孔平均值+0.15
陰性判定：樣品 OD 值< 臨界值（CUT OFF）者為人柯薩奇病毒 A16（Cox-A16）陰性
陽性判定：樣品 OD 值≥ 臨界值（CUT OFF）者為人柯薩奇病毒 A16（Cox-A16）陽性
注意事項
1．操作嚴格按照說明書進行，本試劑不同批號組分不得混用。
2．試劑盒從冷藏環(huán)境中取出應在室溫平衡 15-30 分鐘后方可使用，酶標包被板開封后如未
用完，板條應裝入密封袋中保存。
3．濃洗滌液可能會有結晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結果。
4．封板膜只限一次性使用，以避免交叉污染。
5．底物請避光保存。
6．試驗結果判定必須以酶標儀讀數(shù)為準，使用雙波長檢測時，參考波長為 630nm
7．所有樣品，洗滌液和各種廢棄物都應按傳染物處理。終止液為 2M的硫酸，使用時必須
注意安全。3
保存條件及有效期
1．試劑盒保存：2-8℃。
2．有效期：6 個月4
FOR RESEARCH USE ONLY
Human Coxsachievirus A16
Drug Names
Generic Name：Human Coxsachievirus A16 (Cox-A16) ELISA Kit.
Purpose
This kit allows for the determination of Cox-A16 in Human serum, and
other biological fluids.
Principle of the assay
The kit assay Human Cox-A16 level in the sample，use Purified Human
Cox-A16 antibody to coat microtiter plate wells, make solid-phase antibody,
then add Cox-A16 to wells, Combined With Cox-A16, after washing and
removing non-combinative antigen and other components ,then Combined
Cox-A16 antibody which with HRP labeled, become antibody - antigen -
enzyme- antibody complex, after washing Compley, Add TMB substrate
solution,, TMB substrate becomes blue color At HRP enzyme-catalyzed,
reaction is terminated by the addition of a sulphuric acid solution and the color
change is measured spectrophotometrically at a wavelength of 450 nm.
Compared with the CUTOFF value, according to this to judge Cox-A16 exist in
the sample or not.5
Materials provided with the kit
Materials provided
with the kit
48determinations 96 determinations
Stora
ge
User manual 1 1
Closure plate
membrane
2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8℃
Negative control 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃
Positive control 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃
HRP-Conjugate
reagent
3ml×1 bottle 6ml×1 bottle 2-8℃
Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen Solution
A 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen Solution
B 3ml×1 bottle 6ml×1 bottle 2-8℃
Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8℃
wash solution
（20ml×20 fold）
×1bottle
（20ml×30 fold）
×1bottle
2-8℃
Specimen requirements
1. serum- coagulation at room temperature 10-20 mins， centrifugation 20-min
at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation
appeared, Centrifugal again.
2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20
mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove
supernatant, If precipitation appeared, Centrifugal again.
3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of
2000-3000 r.p.m. remove supernatant, If precipitation appeared,
Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid
Reference to it.
4. cell culture supernatant-detect secretory components, collect sue a6
sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m.
remove supernatant,detect the composition of cells, Dilut cell suspension
with PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeated
freeze-thaw cycles, damage cells and release of intracellular components,
centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,
If precipitation appeared, Centrifugal again.
5. Tissue samples- After cutting samples, check the weight,add PBS
（PH7.2-7.4）, Rapidly frozen with liquid nitrogen, maintain samples at
2-8℃ after melting,add PBS（PH7.4）, Homogenized by hand or Grinders,
centrifugation 20-min at the speed of 2000-3000 r.p.m. remove
supernatant.
6. extract as soon as possible after Specimen collection,and according to the
relevant literature, and should be experiment as soon as possible after the
extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid
repeated freeze-thaw cycles.
7. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP
active.
Assay procedure
1.Number: to sample correspond microtitration well and Number Sequence,
each plate should be set feminine comparison 2 wells, masculine comparison
2 wells, blank comparison 1 well(don’t add sample and HRP-Conjugate
reagent to blank comparison well, other each step the operation are same).
2.add sample：separay add Positive control and Negative control 50μl to the
Positive and Negative well . add Sample dilution 40μl to testing sample well,
then add testing sample 10μl. add sample to the bottom of ELISA plates
coated well , don’t touch the well wall as far as possible, and Gently mix.
3.Incubate: After closing plate with Closure plate membrane ,incubate for 30
min at 37℃.
4.Configurate liquid: 30-fold (or 20-fold)wash solution diluted until 600ml,and7
reserve.
5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing,
add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by
pat.
6.add enzyme： Add HRP-Conjugate reagent 50μlto each well, except the blank
well.
7.incubate：Operation with 3.
8.washing：Operation with 5.
9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each
well, evade the light preservation for 15 min at 37℃
10.Stop the reaction： Add Stop Solution50μl to each well, Stop the reaction(the
blue color change to yellow color).
11. assay：take blank well as zero , Read absorbance at 450nm after Adding
Stop Solution and within 15min.
Determine the result
Test validity: the average of Positive control well≥1.00; the average of
Negative control well ≤0.10.
Calculate Critical(CUT OFF) : Critical= the average of Negative control well
+ 0.15.
Negative control: sample OD< Calculate Critical(CUT OFF) is Cox-A16
Negative control.
Positive control: ample OD≥ Calculate Critical(CUT OFF) is Cox-A16
Positive control.
Important notes
1.Please according to use instruction strictly, Do not mix reagents with those
from other lots.8
2.The kit takes out from the refrigeration environment should be balanced
15-30 minutes in the room temperature then use, ELISA plates coated if
has not use up after opened, the plate should be stored in Sealed bag.
3.washing buffer will Crystallization separation, it can be heated the water
helps dissolve when dilute . Washing does not affect the result.
4.Closure plate membrane only limits the disposable use, in order to avoid
the overlapping pollution
5.The substrate please evade the light preservation.
6.The test result determination must take the microtiter plate reader as a
standard, when use dual-wavelength to assay, Reference wavelength is
630nm.
7.All samples, washing buffer and each kind of reject should according to
infective material process. Stopp Solution is 2M sulphuric acid. You must pay
attention to safe when use .
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.